Lanznaster, D. | Search bioRxiv

bioRxiv2025

scFv intrabody targeting wildtype TDP-43 presents protective effects in a cellular model of TDP-43 proteinopathy

TDP-43 proteinopathies are a set of neurological disorders characterized by the abnormal accumulation and mislocalization of TDP-43 in the cytoplasm, leading to the disruption of the normal function of the protein. In most of the cases, it is the wildtype (wt) form of the protein that is involved. An untargeted high-throughput screen of a single-chain variable fragment (scFv) library was performed using phage display against human full-length wt TDP-43. Two scFvs (B1 and D7) were retained following cellular expression (then termed intrabodies) and colocalization with cytoplasmic TDP-43 in vitro. We generated a 3D structure of full length wt TDP-43 in silico, and used it for epitope mapping. In a cellular model of TDP-43 proteinopathy, D7 enhanced the proteasomal degradation of the insoluble 35-kDa C-terminal fragment TDP-43 and reversed some TDP-43-induced metabolomic alterations, particularly relating to the lipid metabolism. Our findings offer a new scFv intrabody that bind to human wtTDP-43 and modify cellular pathways associated with TDP-43 proteinopathies.

neuroscience↗

bioRxiv2022

Relation between pH alterations in cellular models of amyotrophic lateral sclerosis and TDP-43 protein aggregation

Amyotrophic Lateral Sclerosis (ALS) is the most common neurodegenerative disease affecting motor neurons. The pathophysiology of ALS is not well understood but TDP-43 proteinopathy (aggregation and mislocalization) is one of the major phenomena described. Several factors can influence TDP-43 behavior such as mild pH alterations that can induce conformational changes in recombinant TDP-43, increasing its propensity to aggregate. However to our knowledge, no studies have been conducted yet in a cellular setting, in the context of ALS. We therefore tested the effect of cellular pH alterations on the localization and aggregation of TDP-43. HEK293T cells overexpressing wildtype TDP-43 were incubated for 1h with solutions of different pH (6.4, 7.2, and 8). Incubation of cells for 1h in solutions of pH 6.4 and 8 led to an increase in TDP-43-positive puncta, an effect that was lost after 2h of incubation. This was accompanied by the mislocalization of TDP-43 from the nucleus to the cytoplasm. Our resulats suggest that small alterations in cellular pH affect TDP-43 and increase its mislocalization into cytoplasmic TDP-43-positive puncta, which might suggest a role of TDP-43 in the response of cells to pH alterations.

cell biology↗